黑素细胞中PINK1的表达及在氧化损伤中的作用

Identification of PINK1 Expression in Normal Human Melanocytes and Its Role in Oxidative Damage

目的明确正常人黑素细胞PIG1中PINK1的表达,探讨PINK1在黑素细胞氧化损伤中的作用。方法 (1)采用逆转录实时定量PCR(RT-qPCR)及免疫印迹法(WB),检测PIG1黑素细胞中PINK1的mRNA及蛋白的表达水平;(2)不同浓度H2_O_2处理PIG1细胞24h,CCK-8法测定细胞增殖活力,确定H_2O_2最适浓度;(3)设计并合成3对PINK1干扰片段(siRNA)转染PIG1细胞,采用RT-qPCR法筛选干扰效率最高的siRNA;(4)实验分组:对照组、Mock组、NC组、转染组。细胞转染48h后,再加入H_2O_2处理24h,观察各组细胞形态,CCK-8法测定细胞增殖活力。结果 (1)在PIG1黑素细胞中检测到PINK1 mRNA及蛋白的表达;(2)黑素细胞增殖活力呈H2O2浓度依赖性降低,0.6mmol/L H_2O_2组降低至(49.02±2.40)%,接近IC50,故以此浓度作用24h建立氧化损伤模型;(3)与Mock组相比,PINK1-siRNA3转染48h干扰效率最高(>90%);(4)与对照组相比,PINK1-siRNA3转染组树突回缩及变圆悬浮的细胞明显增多,转染组细胞增殖活力明显降低(P<0.01)。结论本研究首次明确了PINK1在正常人黑素细胞中的表达,下调PINK1可加重H_2O_2所致的黑素细胞形态学改变及活力降低,表明PINK1对黑素细胞具有一定的保护性作用。

Objective To detect the expression of PINK1 in normal human PIG1 melanocytes and to explore the role of PINK1 in oxidative damage of melanocytesMethods RT-qPCR and WB analyses were performed respectively to detect mRNA and protein expression of PINK1 in PIG1 melanocytesAfter treatment with various concentrations of H2O2 for 24h,proliferation activity of PIG1 melanocytes was determined by CCK-8 method,to pinpoint the optimal concentration of H2O2Three pairs of PINK1 interfering RNAs（siRNAs） that were designed and synthesized were transfected into PIG1 cellsRT-qPCR was used to screen the siRNA with the highest interference efficiencyThe experimental group consisted of control group,Mock group,NC group and transfection groupAfter 48h of cell transfection,H2O2 was added for incubation for 24h to observe cell morphology in each group,and CCK-8 assay was used to determine cell proliferation activityResults The mRNA and protein expression of PINK1 was identified in PIG1 melanocytes by RT-qPCR and WBThe proliferation activity of melanocytes was decreased in an H2O2 concentration-dependent manner to（4902±240）% in 06mmol/L H2O2 group,which was close to IC50Therefore,this concentration was used for incubation for 24h to establish an oxidative damage modelCompared with Mock group,PINK1-siRNA3 had the highest interference efficiency（〉90%） at 48h after transfectionCompared with the control group,dendritic retraction and round-suspended cells were significantly increased,whereas the proliferation activity was significantly decreased in the PINK1-siRNA3 transfection group（P〈001）ConclusionIt is for the first time that the expression of PINK1 is identified in normal human melanocytes in our studyDown-regulation of PINK1 aggravates the morphological changes and decreased viability of H2O2-treated melanocytes,indicating that PINK1 has a protective effect on melanocytes